The present invention relates generally to the modification and aging of proteins through reaction with glucose and other reducing sugars, such as fructose or ribose and more particularly to the inhibition of nonenzymatic glycation of proteins which often results in formation of advanced glycation endproducts and crosslinks.
An elevated concentration of reducing sugars in the blood and in the intracellular environment results in the nonenzymatic formation of glycation and dehydration condensation complexes known as advanced glycation end-products (AGE""s). These complex products form on free amino groups on proteins, on lipids and on DNA (Bucala and Cerami, 1992; Bucala et al., 1993; Bucala et al., 1984). This phenomenon is called xe2x80x9cbrowningxe2x80x9d or xe2x80x9cMaillardxe2x80x9d reaction and was discovered early in this century by the food industry (Maillard, 1916). The significance of a similar process in biology became evident only after the discovery of the glycosylated hemoglobins and their increased presence in diabetic patients (Rahbar, 1968; Rahbar et al., 1969). In human diabetic patients and in animal models of diabetes, these nonenzymatic reactions are accelerated and cause increased AGE formation and increased glycation of long-lived proteins such as collagen, fibronectin, tubulin, lens crystallin, myelin, laminin and actin, in addition to hemoglobin and albumin, and also of LDL associated lipids and apoprotein. Moreover, brown pigments with spectral and fluorescent properties similar to those of late-stage Maillard products have also been found in vivo in association with several long-lived proteins such as lens crystallin proteins and collagen from aged individuals. An age-related linear increase in pigments was observed in human dura collagen between the ages of 20 to 90 years. AGE modified proteins increase slowly with aging and are thought to contripbute to normal tissue remodeling. Their level increases markedly in diabetic patients as a result of sustained high blood sugar levels and lead to tissue damage through a variety of mechanisms including alteration of tissue protein structure and function, stimulation of cellular responses through AGE specific receptors or the generation of reactive oxygen species (ROS) (for a recent review see Boel et al., 1995). The structural and functional integrity of the affected molecules, which often have major roles in cellular functions, become perturbed by these modifications, with severe consequences on affected organs such as kidney, eye, nerve, and micro-vascular functions (Silbiger et al., 1993; Brownlee et al., 1985).
Structural changes on macromolecules by AGE""s are known to accumulate under normal circumstances with increasing age. This accumulation is severely accelerated by diabetes and is strongly associated with hyperglycemia. For example, formation of AGE on protein in the subendothelial basement membrane causes extensive cross-link formation which leads to severe structural and functional changes in protein/protein and protein/cell interaction in the vascular wall (Haitoglou et al., 1992; Airaksinen et al., 1993).
Enhanced formation and accumulation of advanced glycation end products (AGE""s) have been proposed to play a major role in the pathogenesis of diabetic complications and in aging, leading to progressive and irreversible intermolecular protein crosslinkings (Monnier et al., 1986). This process is accelerated by diabetes and has been postulated to contribute to the development of a range of diabetic complications including nephropathy (Nicholls and Mandel, 1989), retinopathy (Hammes et al., 1991) and neuropathy (Cameron et al., 1992). Particularly, tissue damage to the kidney by AGE""s leads to progressive decline in renal function and end-stage renal disease (ESRD) (Makita et al., 1994), and accumulation of low-molecular-weight (LMW) AGE peptides (glycotoxins) (Koschinsky et al., 1997) in the serum of patients with ESRD (Makita et al., 1991). These low molecular weight (LMW)-AGE""s can readily form new crosslinks with plasma or tissue components, e.g., low density lipoprotein (LDL) (Bucala et al., 1994) or collagen (Miyata et al., 1993) and accelerate the progression of tissue damage and morbidity in diabetics.
The Diabetic Control and Complications Trial (DCCT), has identified hyperglycemia as the main risk-factor for the development of diabetic complications (The Diabetes Control and Complications Trial Research Group, 1993). Although there is no consensus regarding the pathogenic link between hyperglycemia and diabetic complications, formation of advanced glycation endproducts (AGE""s) has been implicated as a major pathogenic process in the long-term complications of diabetes, namely nephropathy, neuropathy and retinopathy.
Particularly, tissue damage to the kidney by AGE""s leads to progressive decline in renal function and end-stage renal failure (ESRD) (Makita et al., 1994), and accumulation of low-molecular-weight (LMW) AGE peptides (glycotoxins) (Koschinsky et al., 1997) in the serum of patients with ESRD (Makita et al., 1991). These low molecular weight (LMW)-AGE""s can readily form new cross-links with plasma or tissue components, e.g., low density lipoprotein (LDL) (Bucala et al., 1994) or collagen (Baily et al., 1998) and accelerate the progression of tissue damage and morbidity in diabetes.
Although the mechanism of non-enzymatic glycation is not completely understood and the precise structures of major AGE components remain to be established, xcex1-dicarbonyl compounds have been identified as major intermediates in the glycation pathways and cross-linking of proteins by glucose (Baynes and Thorpe, 1999). Hyperglycemia is associated with the formation of dicarbonyl compounds, particularly methylglyoxal (MGO), glyoxal (GO), 3-deoxyglucosone (3-DG), glycoaldehyde and dehydroascorbate (Onorato et al., 1998). In patients with both insulin-dependent and non-insulin dependent diabetes, the concentration of MGO was found to be increased 2-6-fold (McLellan et al., 1994). The major source of MGO is thought to be the non-enzymatic dephosphorylation of the triosedihydroxyacetone phosphate and glyceraldehyde-3-phosphate. In addition, glucose auto-oxidation and degradation of Amadori products and enzymatic catabolism of acetone through the acetol pathway (Phillips and Thornalley, 1993) are alternative sources of MGO formation. The glyoxylase system (I and II) and aldose reductase catalyze the detoxification of MGO to D-lactate. MGO binds to and irreversibly modifies arginine and lysine residues in proteins. MGO modified proteins have been shown to be ligands for the AGE receptor (Beisswenger et al., 1998), indicating that MGO modified proteins are analogous (Beisswenger et al., 1998) to those found in AGE""s. Most recently, the effects of MGO on LDL has been characterized in vivo and in vitro (Westwood et al., 1997).
Lipid peroxidation of polyunsaturated fatty acids (PUFA), such as arachidonate, also yield carbonyl compounds; some are identical to those formed from carbohydrates (Schalkwijk et al., 1998), such as MGO and GO, and others are characteristic of lipid, such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE) (Bucala et al., 1993). The latter carbonyl compounds produce lipoxidation products, such as MDA-lysine, lysine-MDA-lysine, HNE-protein adducts (HNE Lys, His, Cys) (Al-Abed et al., 1996). AGE compounds characterized in vitro and in vivo are either the result of glycation alone or glycation-oxidation modification called glycoxidation products. Glycoxidation products that have been detected in tissue proteins are Nxcex5-carboxymethyl-lysine (CML), Nxcex5-carboxyethyl-lysine (CEL), Nxcex5-carboxymethyl-ethanolamine (CME) (Requena et al., 1997), pentosidine, glyoxal-lysine-dimer (GOLD) and methylglyoxal-lysine-dimer (MOLD). These products may be derived from either glycoxidation or lipoxidation reactions. Other AGE""s detected in tissue proteins are pyrraline, crossline imidazolium salts formed by the reaction of MOLD or GOLD and imidazolones formed by the reaction of MGO or 3-DG with arginine. Most of the above AGE""s are involved in cross-linking of proteins. Furthermore, MGO has been found not only as the most reactive dicarbonyl AGE-intermediate in cross-linking of proteins, a recent report has found MGO to generate reactive oxygen species (ROS) (free radicals) in the course of glycation reactions (Yim et al., 1995). Indeed, three types of free radical species were produced and their structures were determined by EPR spectroscopy. The superoxide radical anion was produced only in the presence of oxygen in these reactions, more evidence that ROS are generated during glycation reaction and AGE-formation.
These findings and other reports provided evidence that indeed oxidative stress and AGE-formation are undividedly intertwined. Oxidative stress is apparent in pathology associated with aging and common end points of chronic diseases, such as atherosclerosis, diabetes, rheumatoid arthritis and uremia (Baynes and Thorpe, 1999). What is not clear is whether oxidative stress has a primary role in the pathogenesis of diabetic complications or whether it is a secondary indicator of end-stage tissue damage in diabetes (Baynes and Thorpe, 1999). The increase in glycoxidation and lipoxidation products in plasma and tissue proteins suggests that oxidative stress is in fact increased in diabetes (Baynes, 1996). The issue is whether oxidation stress occurs at early stage in diabetes, preceding the appearance of complications, or whether it is merely a common consequence of the tissue damage, reflecting the presence of complications. Several lines of evidence indicated that one of the critical pathogenic consequences of hyperglycemia in diabetes is a deficit in detoxification of reactive carbonyl (dicarbonyl) compounds. The increase in reactive carbonyls derived from both oxidative and nonoxidative reactions (defined as carbonyl stress) leads to increased chemical modification of proteins, and then, at a late stage, to oxidant stress and tissue damage. Thus, intervention should begin at the level of carbonyl stress, long before the appearance of overt oxidative stress and damage (Baynes and Thorpe, 1999).
Direct evidence indicating the contribution of AGE""s in the progression of diabetic nephropathy has recently been reported (Vlassara et al., 1994). Indeed, the infusion of pre-formed AGE""s into healthy rats induces glomerular hypertrophy and mesangial sclerosis, gene expression of matrix proteins and production of growth factors (Brownlee et al., 1991; Vlassara et al., 1995). Further studies have revealed that aminoguanidine (AG), an inhibitor of AGE formation, ameliorates tissue impairment of glomeruli and reduces albuminuria in induced diabetic rats (Soulis-Liparota et al., 1991; Itakura et al., 1991). In humans, decreased levels of hemoglobin (Hb)-AGE (Makita et al., 1992) concomitant with amelioration of kidney function as the result of aminoguanidine therapy in diabetic patients, provided more evidence for the importance of AGE""s in the pathogenesis of diabetic complications (Bucala and Vlassara, 1997).
The global prevalence of diabetes mellitus, in particular in the United States, afflicting millions of individuals with significant increases of morbidity and mortality, together with the great financial burden for the treatment of diabetic complications in this country, are major incentives to search for and develop drugs with a potential of preventing or treating complications of the disease. So far the mechanisms of hyperglycemia-induced tissue damage in diabetes are not well understood. However, four pathogenic mechanisms have been proposed, including increased polyol pathway activity, activation of specific protein kinase C (PKC) isoforms, formation and accumulation of advanced glycation endproducts, and increased generation of reactive oxygen species (ROS) (Kennedy and Lyons, 1997). Most recent immunohistochemical studies on different tissues from kidneys obtained from ESRD patients (Horie et al., 1997) and diabetic rat lenses (Matsumoto et al., 1997), by using specific antibodies against carboxymethyllysine (CML), pentosidine, the two known glycoxidation products and pyrraline, have localized these AGE components in different lesions of the kidneys and the rat lens, and have provided more evidence in favor of protein-AGE formation in close association with generation of ROS to be major factors in causing permanent and irreversible modification of tissue proteins. Therefore, inhibitors of AGE formation and antioxidants hold promise as effective means of prevention and treatment of diabetic complications.
In addition to aging and diabetes, the formation of AGEs has been linked with several other pathological conditions. IgM anti-IgG-AGE appears to be associated with clinical measurements of rheumatoid arthritis activity (Lucey et al., 2000). A correlation between AGEs and rheumatoid arthritis was also made in North American Indians (Newkirk et al., 1998). AGEs are present in brain plaques in Alzheimer""s disease and the presence of AGEs may help promote the development of Alzheimer""s disease (Durany et al., 1999; Munch et al., 1998; Munch et al., 1997). Uremic patients have elevated levels of serum AGEs compared to age-matched controls (Odani et al., 1999; Dawnay and Millar, 1998). AGEs have also been correlated with neurotoxicity (Kikuchi et al., 1999). AGE proteins have been associated with atherosclerosis in mice (Sano et al., 1999) and with atherosclerosis in persons undergoing hemodialysis (Takayama et al., 1998). A study in which aminoguanidine was fed to rabbits showed that increasing amounts of aminoguanidine led to reduced plaque formation in the aorta thus suggesting that advanced glycation may participate in atherogenesis and raising the possibility that inhibitors of advanced glycation may retard the process (Panagiotopoulos et al., 1998). Significant deposition of N(epsilon)-carboxymethyl lysine (CML), an advanced glycation endproduct, is seen in astrocytic hyaline inclusions in persons with familial amyotrophic lateral sclerosis but is not seen in normal control samples (Kato et al., 1999; Shibata et al., 1999). Cigarette smoking has also been linked to increased accumulation of AGEs on plasma low density lipoprotein, structural proteins in the vascular wall, and the lens proteins of the eye, with some of these effects possibly leading to pathogenesis of atherosclerosis and other diseases associated with tobacco usage (Nicholl and Bucala, 1998). Finally, a study in which aminoguanidine was fed to rats showed that the treatment protected against progressive cardiovascular and renal decline (Li et al., 1996).
The mechanism of the inhibitory effects of aminoguanidine has been investigated and it has been demonstrated that AG acts as a general-carbonyl scavenger, trapping both oxidative and nonoxidative AGE precursors as well as intermediates in lipid peroxidation reactions (Chen and Cerami, 1993). In vitro and in vivo studies indicated the AG reacts with MGO to produce substituted triazines (Chen and Cerami, 1993). Most recently, metformin, one of the drugs used for many years in the treatment of type 2 diabetes, was demonstrated to inactivate and reduce MGO levels both in vivo and in vitro (Beisswenger et al., 1999), indicating the inhibitory function of metformin in AGE-formation. Despite the limitations of our current knowledge, there is leading evidence that trapping of reactive carbonyl compounds, the chemical intermediates between hyperglycemia/hyperlipidemia and diabetic complications, may be a valuable strategy for inhibiting or delaying diabetic complications (Baynes and Thorpe, 1999). Considering the diversities of these carbonyl intermediates, different drugs with diverse structures could be discovered as inhibitors of carbonyl compounds.
Several other potential drug candidates as AGE inhibitors have been reported recently. These studies evaluated the agent""s ability to inhibit AGE formation and AGE-protein crosslinking compared to that of aminoguanidine (AG) through in vitro and in vivo evaluations (Nakamura et al., 1997; Kochakian et al., 1996). A recent breakthrough in this field is the discovery of a compound, N-phenacylthiazolium bromide (PTB), which selectively cleaves AGE-derived protein crosslinks in vitro and in vivo (Vasan et al., 1996; Ulrich and Zhang, 1997). The pharmacological ability to break irreversible AGE-mediated protein crosslinking offers potential therapeutic use.
It is well documented that early pharmaceutical intervention against the long-term consequences of hyperglycemia-induced crosslinking prevent the development of severe late complications of diabetes. The development of nontoxic and highly effective drugs that completely stop glucose-mediated crosslinking in the tissues and body fluids is a highly desirable goal. The prototype of the pharmaceutical compounds investigated both in vitro and in vivo to intervene with the formation of AGE""s on proteins is aminoguanidine (AG), a small hydrazine-like compound (Brownlee et al., 1986). However, a number of other compounds were found to have such an inhibitory effect on AGE formation. Examples are D-lysine (Sensi et al., 1993), desferrioxamine (Takagi et al., 1995), D-penicillamine (McPherson et al., 1988), thiamine pyrophosphate and pyridoxamine (Booth et al., 1997) which have no structural similarities to aminoguanidine.
Clinical trials of AG as the first drug candidate intended to inhibit AGE formation are in progress (Corbett et al., 1992). A number of hydrazine-like and non-hydrazine compounds have been investigated. So far AG has been found to be the most useful with fewer side effects than other tested compounds of the prior art. However, AG is a well known selective inhibitor of nitric oxide (NO) and can also have antioxidant effects (Tilton et al., 1993).
A number of other potential drug candidates to be used as AGE inhibitors have been discovered recently and evaluated both in vitro and in vivo (Nakamura et al., 1997; Soulis et al., 1997). While the success in studies with aminoguanidine and similar compounds is promising, the need to develop additional inhibitors of AGE""s continues to exist in order to broaden the availability and the scope of this activity and therapeutic utility.
Pentoxifylline, pioglitazone and metformin have been found to inhibit the nonenzymatic glycation of proteins which often results in formation of advanced glycation endproducts and crosslinks. The nonenzymatic glycation and crosslinking of proteins is a part of the aging process with the glycation endproducts and crosslinking of long-lived proteins increasing with age. This process is increased at elevated concentrations of reducing sugars in the blood and in the intracellular environment such as occurs with diabetes. The structural and functional integrity of the affected molecules become perturbed by these modifications and can result in severe consequences. The compounds of the present invention can be used to inhibit this process of nonenzymatic glycation and crosslinking and therefore to inhibit some of the ill effects caused by diabetes or by aging. The compounds are also useful for preventing premature aging, rheumatoid arthritis, Alzheimer""s disease, uremia, neurotoxicity, atherosclerosis and spoilage of proteins in food and can prevent discoloration of teeth.